ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide binding proteins that were originally identified by their ability to stimulate in vitro cholera toxin (CT)-catalyzed ADP- ribosylation of Gs alpha, the alpha subunit of the stimulatory heterotrimeric GTP-binding protein of the adenylyl cyclase system. ARFs have been implicated in intracellular protein trafficking and are thought to be involved in the assembly of COP-coated vesicles. Most recently, ARFs have been shown to stimulate an isoform of phospholipase D (PLD) and thus may also have a role in intracellular signalling. Based on molecular cloning, at least six mammalian ARFs are known. These fall into three classes based on deduced amino acid sequence, size, and phylogenetic analysis. Molecular cloning has also allowed the identification of ARF-like proteins (ARLs), that are ~40 - 50% identical to ARF family members and have not demonstrated any of the activities associated with ARF. In defining roles for the ARF classes, experimental evidence showed that while members of all three ARF classes stimulated both CTA and PLD, human ARL1 failed to stimulate either. Examination of the effects of amino-terminal myristoylation on PLD activation demonstrated that myristoylation clearly enhanced ARF stimulation of PLD and appeared to slightly reverse the inactivity of ARL towards PLD. ARL's inactivity has allowed its use in the construction of chimeric proteins with ARF for use in defining sequence elements of ARF important for the activation of CT and PLD. Chimeric molecules constructed from ARF and ARL have demonstrated that an ARF domain required for the activation of CT is carboxy-terminal to position 73, whereas a domain responsible for the activation of PLD is amino- terminal to position 73. The data are in agreement with studies, using an amino-terminal deletion mutant, which suggest that the amino terminus is not required for the activation of CT, but is for activation of PLD. Further definition of ARF domains is critical to identifying the structural basis for the diverse functions of ARF.